DNA modifications in the form of 5-methylcytosine (5mC) and the recently identified 5-hydroxymethylcytosine (5hmC) represent the two major epigenetic marks found in mammalian genome and they impact a broad range of biological processes from gene regulation to normal development. Detecting aberrant 5mC and 5hmC changes in the cell-free DNA (cfDNA) may represent an attractive noninvasive approach for cancer diagnostics. cfDNA is the circulating DNA found in our blood originated from different tissues and has been utilized for noninvasive prenatal tests, organ transplant diagnostics, and cancer detection. Compared the intensive research on cell-free 5mC DNA as a biomarker for cancer diagnostics, cell-free 5hmC DNA has remain unexploited, mostly due to the low level of 5hmC compared to 5mC in the human genome (10 to 100-fold less than 5mC) and the lack of a sensitive low-input 5hmC DNA sequencing method to work with the minute amounts of cfDNA (typically only a few nanograms per ml of plasma).